P749: THE ROLE OF INCREASED IRF1 EXPRESSION OF NATURAL KILLER CELLS IN THE IMMUNE PATHOGENESIS OF SEVERE APLASTIC ANAEMIA
Authors: Liu Zixuan, Zhang Tian, Liu Chunyan, Rong Fu
Journal: HemaSphere (2023)

Topic: 11. Bone marrow failure syndromes incl. PNH - Biology & Translational Research Background: Natural killer (NK) cells play an essential immunoregulatory role in the pathogenesis of severe aplastic anaemia (SAA). NK cells exert a regulatory effect on adaptive immunity by secreting interferon (IFN) and stimulating cytotoxic T cell proliferation and activation. Our previous research showed that IFN regulatory factor 1 (IRF-1) was essential in developing NK cells. Moreover, an increased expression of IRF-1 was detected in NK cells, which was associated with NK cell dysfunction and the pathogenesis of SAA. Aims: In this study, the expression of IRF-1 was evaluated in the bone marrow NK cells of patients with SAA and murine model to further understand the pathogenesis of SAA. Methods: The expression of IRF-1 in NK cells from the bone marrow was detected by flow cytometry. The expression levels of IRF-1 in NK cells were detected using Rt-PCR and western blot.NK92MI cells were selected to induce IRF-1 overexpression in NK cells using IFN-α or to reduce IRF-1 expression using the IRF-1 inhibitor rottlerin. Fluorescence-activated cell sorting was used to detect the activation states of NKG2A and NKG2D in NK cells. CCK-8 was used to detect NK cell proliferation, while annexin V was used to detect NK cell apoptosis. The treated NK cells were co-cultured with CD8+T cells. Then, a cytometric bead array system was used to detect the levels of TNF-α, IFN-γ, IL-10, and IL-12 in vivo. The functions and apoptosis rates of the CD8+T cells cultured with the treated NK cells were measured. The murine model of AA was established,following IRF-1 inhibitor treatment, the probability of survival, general condition, blood routine, and bone marrow count of the mice in each group were compared. Results: The NK cells of the SAA initial treatment group had a higher expression of IRF-1 compared to the remission and control groups. IRF-1 expression in NK cells of SAA patients was negatively correlated with the peripheral blood platelet count as well as the proportion of neutrophils and reticulocytes,but positively correlated with the proportion of lymphocytes (P <0.05). The expression of NKG2A on the surface of NK92MI cells decreased. Meanwhile, the expression of NKG2D increased. Adding the IRF-1 blocker promoted NK-92MI cell proliferation and inhibited apoptosis. The levels of TNF-α, IFN-γ, IL-10, and IL-12 in the supernatant were significantly reduced after the addition of the IRF-1 blocker. The apoptosis rate of the CD8+T cells was increased. The level of signalling pathway protein NF-κB p65 in the NK cells in the IRF-1 inhibitor group was lower than that of the control group.The NK cells of the SAA mice had a higher IRF-1 expression than the TBI mice and normal controls. On the 17th day of model establishment, the weight, haemogram, and bone marrow cell count of the AA mice were lower than those of the NC group (P<0.05). The combination of the CsA and IRF-1 inhibitor infusion had a more significant therapeutic effect than CsA treatment alone. IRF-1 inhibitor infusion therapy possibly had a synergistic effect with CsA. Conclusion: The findings of this study confirmed that increased IRF-1 expression inhibited NK cells, thus contributing to the pathogenesis of SAA. Following IRF-1 inhibitor reinfusion, the probability of survival, general condition, blood cell count, and bone marrow cell count of the SAA mice improved. Additionally, the combined treatment with CsA increased the effectiveness of the treatment. The results of this study helped enhance the understanding of the pathogenesis of SAA, and new treatment targets for improving the efficacy of SAA treatment were identified.Keywords: Murine models, Aplastic anemia, Interferon regulatory factor, NK cell